Anti-Inflammatory and Anti-Thrombotic Effects of the Fungal Metabolite Galiellalactone in Apolipoprotein E-Deficient Mice

RESEARCH ARTICLE

Anti-Inflammatory and Anti-ThromboticEffects of the Fungal MetaboliteGaliellalactone in Apolipoprotein E-DeficientMiceFranziska Bollmann1,2☯, Sven Jäckel2☯, Lisa Schmidtke1, Katharina Schrick1,Christoph Reinhardt2, Kerstin Jurk2, ZhixiongWu1, Ning Xia1, Huige Li1, Gerhard Erkel3,UlrichWalter2, Hartmut Kleinert1‡*, Andrea Pautz1‡

1 Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz,Mainz, Germany, 2 Center for Thrombosis and Hemostasis (CTH), University Medical Center of theJohannes Gutenberg University Mainz, Mainz, Germany, 3 Department of Molecular Biotechnology andSystems Biology, Technical University Kaiserslautern, Kaiserslautern, Germany

☯ These authors contributed equally to this work.‡ AP and HK are co-senior authors.* [email protected]

AbstractPatients suffering from chronic inflammatory diseases have an increased mortality risk re-

sulting from cardiovascular disorders due to enhanced atherosclerotic and thrombotic

events. Until now, it is not completely understood in which way an abnormal expression of

pro-inflammatory mediators contributes to this elevated cardiovascular risk, but there is a

need for new drugs that on the one hand suppress the expression of pro-inflammatory medi-

ators and on the other hand inhibit arterial platelet adhesion. Thus, we analyzed the anti-in-

flammatory and anti-thrombotic capacity of the fungal metabolite Galiellalactone in

atherosclerosis-prone apolipoprotein E-deficient mice. Treatment of the mice with Galiella-

lactone lowered the inflammatory expression profile and improved blood clotting times, as

well as platelet adhesion to the injured common carotid artery. The results indicate that ad-

ministration of Galiellalactone is able to reduce the extent of inflammation and arterial plate-

let adhesion in this mouse model.

IntroductionAtherosclerosis as well as thrombotic events as its clinical manifestation results in increasedcardiovascular death (about 50%) of people suffering from chronic inflammatory diseases,such as rheumatoid arthritis [1–4] or inflammatory bowel disease [5]. This increased mortalityrate is independent of classic risk factors, as hypercholesterolemia, age, gender, or smoking [6].Nowadays, the key role of inflammation during the progression of atherosclerosis is well ac-cepted [7, 8]. Since pro-inflammatory mediators as tumor necrosis factor-α (TNFα) or

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Citation: Bollmann F, Jäckel S, Schmidtke L, SchrickK, Reinhardt C, Jurk K, et al. (2015) Anti-Inflammatory and Anti-Thrombotic Effects of theFungal Metabolite Galiellalactone in Apolipoprotein E-Deficient Mice. PLoS ONE 10(6): e0130401.doi:10.1371/journal.pone.0130401

Academic Editor: Kathleen Freson, University ofLeuven, BELGIUM

Received: February 13, 2015

Accepted: May 20, 2015

Published: June 15, 2015

Copyright: © 2015 Bollmann et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.

Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.

Funding: Funding provided by Federal Ministry ofEducation and Research BMBF 01EO1003 http://www.bmbf.de/ (UW); Innovation Foundation of theState of Rhineland-Palatinate 961-386261/917Khttp://stiftung-innovation.rlp.de/ (HK); InnovationFoundation of the State of Rhineland-Palatinate 961-386261/1105AP http://stiftung-innovation.rlp.de/ (AP);DFG LI 1759/1-1 http://www.dfg.de/ (HK); DFG RE3450/3-1 http://www.dfg.de/ (CR); DFG PA 1933/2-3http://www.dfg.de/ (AP).

Interleukin-1β (IL1β) are elevated in rheumatoid arthritis patients and trigger atherogenesis[9], the design of new drugs is focusing on the reduction of these inflammatory mediators. Butso far, only contrary studies exist, demonstrating either beneficial or no beneficial effects of ananti-TNFα therapy on the cardiovascular risk of rheumatoid arthritis patients [10, 11]. Cur-rently, there is a need for new drugs, which suppress vascular inflammation signaling to reduceatherosclerosis development and arterial thrombus formation.

Therefore, we investigated whether the fungal metabolite Galiellalactone (Gal) impairs inflam-mation and thrombosis in a model of Apolipoprotein E (ApoE)-deficient mice. In previous studiesGal was shown to be an anti-inflammatory compound that inhibits Interleukin-6 (IL6) signalingin a Janus Kinase/Signal Transducer and Activator of Transcription (STAT)—dependent mannerand reduces experimental asthma [12, 13]. In addition, Gal has been described as a direct inhibitorof the transcription factor STAT3 [14] and to interfere with the nuclear import of the transcriptionfactor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)[15].

For the first time, we now analyzed the anti-thrombotic capacity of this fungal metabolitewith respect to inflammatory marker gene expression, blood clotting times, and in vivo plateletadhesion to injured carotid artery in mice.

Our results indicate a role for Gal in suppressing the expression of pro-inflammatory genesin the aorta, as well as inhibitory effects of Gal on the coagulation system and on platelet adhe-sion to the Arteria carotis communis.

Materials and Methods

AnimalsApoE-deficient mice (ApoE-ko) on a C57BL/6 background (stock 002052; The Jackson Labo-ratory) were housed in accordance with standard animal care requirements in groups up tofive animals per cage. All mice were maintained under specific pathogen-free conditions on a12/12 hours light/dark circle. Water and food were given ad libitum. Genotyping of the animalswas performed by polymerase chain reaction, using primers that span the regions of the wildtype genes disrupted by the targeting vectors. The following oligonucleotides (all obtainedfrom Sigma-Aldrich; Hamburg, Germany) were used for genotyping the ApoE locus using po-lymerase chain reactions: ApoE-wt/for GCC TAG CCG AGG GAG AGC CG; ApoE-wt-rev TGTGAC TTG GGA GCT CTG CAG C; ApoE-ko-rev GCC GCC CCG ACT GCA TCT.

Experimental mice at the age of 6 weeks were either fed with normal chow diet (ND) orwestern-type diet (WD) for 18 weeks. For the last six weeks of this period, the WD-fed micewere treated every other day with an intra peritoneal injection of either the fungal metaboliteGaliellalactone (Gal; 10 mg/kg) or vehicle (PBS + 10% ethanol; PBS/EtOH). All experimentswere performed between 8 a.m. and 8 p.m. in the laboratory.

All procedures performed on mice were approved by the Institutional Animal Care and UseCommittee (“Landesuntersuchungsamt” State of Rhineland-Palatinate, Germany; 23177-07/G11-1-010 and 23177-07/G11-1-018) in accordance with the German animal protection law andthe guidelines for the use of experimental animals as stipulated by the Guide of Care and Use ofLaboratory Animals of the National Institutes of Health. Mice were euthanized by intra peritone-al injection of 700 μL pentobarbital solution (1% pentobarbital in PBS), as not stated otherwise.

Quantitative Real-Time Reverse Transcription Polymerase ChainReaction AnalysesTo analyze the gene expression, the RNA of aortas as well as livers from experimental animalswas isolated. Therefore, the aortas or livers were homogenized in guanidinium thiocyanate

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Competing Interests: The authors have declaredthat no competing interests exist.

buffer and total RNA was isolated by guanidinium thiocyanate/phenol/chloroform extractionas previously described [16].

For mRNA expression analyses, two-step real-time RT PCRs (qPCRs) using either TaqManprobes [17] or SYBR Green as described before [18] were performed. 500 ng total RNA was re-verse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosys-tems; Darmstadt, Germany) following the manufacturer’s recommendations. Subsequently theqPCR reaction was performed using the following oligonucleotides (obtained from Sigma-Al-drich; Hamburg, Germany) as sense and antisense primers, as well as TaqMan hybridizationprobes (5’-6FAM; 3’-TAMRA) (Table 1).

mRNA expression data were normalized either to Glyceraldehyde-3-phosphate dehydroge-nase or DNA polymerase IIa mRNA expression. To calculate the relative mRNA expressions,the 2(-ΔΔCt) method [19] was used.

Coagulation AnalysesFor coagulation analyses, freshly collected blood was incubated with 1/10 v/v citrate solution(Sigma-Aldrich; Hamburg, Germany) to inhibit blood coagulation. Afterwards, the blood wascentrifuged for 20 minutes at 600 xg, the obtained plasma was diluted ¼ with PBS, and usedfor blood coagulation assays, using a KC4 Delta Coagulometer (Tcoag; Bray, Ireland).

To determine the prothrombin time (PT), 50 μL plasma was incubated for 1 minute at37°C. After adding 100 μL thromborel S (Siemens Healthcare; Marburg, Germany) the timeuntil complete clotting of the sample occurred was measured.

For analyzing the partial thromboplastin time (aPTT), 50 μL plasma was incubated with50 μL pathromtin SL (Siemens Healthcare; Marburg, Germany) for 5 minutes at 37°C. Thereaction was started after adding 50 μL of a 0.025 M calcium chloride solution (SiemensHealthcare; Marburg, Germany). The time until complete clotting of the sample occurred wasmeasured.

Hematologic AnalysisCitrated whole mouse blood was collected by intracardiac puncture. Platelet counts, whiteblood cell counts, and red blood cell counts were determined using an automatic cell counter(KX-21; Sysmex, Kobe, Japan).

Mouse Thrombosis Model in the Common Carotid ArteryExperimental ApoE-deficient mice fed with ND or WD (treated with Gal or vehicle) were anes-thetized by intra peritoneal injection of a solution of 5 mg/kg midazolame (RatiopharmGmbH; Ulm, Germany), 0.5 mg/kg medetomidine (Pfizer Deutschland GmbH; Berlin, Ger-many), and 0.05 mg/kg fentanyl (Janssen-Cilag GmbH; Neuss, Germany), as described previ-ously [20]. The animals were fixed on a custom-built stage and maintained at physiologicaltemperature. As a model of arterial thrombosis, carotid injury was induced as previously de-scribed [21]. Therefore, a polyethylene catheter (0.28 mm ID, 0.61 mmOD; Smiths MedicalDeutschland GmbH; Grasbrunn, Germany) was implanted into the right jugular vein. The leftcommon carotid artery was dissected free and ligated vigorously (7.0 monofil polypropylenefilament, Prolene; Ethicon; Norderstedt, Germany) near the carotid bifurcation for 5 min to in-duce vascular injury. Before and after vascular injury, the fluorescent platelets were visualizedin situ by intra-vital epifluorescence high-speed video microscopy of the left common carotidartery. All mice with bleedings or any injury of the carotid artery during surgery were excludedfrom further analysis. There was no difference in the exclusion rate across the different experi-mental groups. All animals were euthanized by cervical dislocation.

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Table 1. Oligonucleotides as sense and antisense primers and TaqMan hybridization probes.

CCL2/MCP1 Chemokine C-C motif ligand 2

Sense AGG TCC CTG TCA TGC TTC TG

Antisense TCA TTG GGA TCA TCT TGCT G

CTSS Cathepsin S

Sense CAT GGT GTT CTT GTG GTT GG

Antisense CAA TAA CTA GCA ATT CCG CAG TG

Probe CTG GCT TGT GAA AAA CAG TTG GGG C

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

Sense TTC ACC ACC ATG GAG AAG GC

Antisense GGC ATG GAC TGT GGT CAT GA

Probe TGC ATC CTG CAC CAC CAA CTG CTT AG

FII Coagulation factor II

Sense CAG CTA TGA GGA GGC CTT TG

Antisense TCA CAC CCA GAT CCA TAG CA

FVIII Coagulation factor VIII

Sense TGC CTG ACC CGC TAT TAT TC

Antisense AGC GTT GCA TGT TCT CTG TG

FX Coagulation factor X

Sense TCA GCC TGC TCT GTG TTG TC

Antisense TCG AAG ATT TCA CGG ACCTC

IL1β Interleukin-1β

Sense CAA CCA ACA AGT GAT ATT CTC CAT G

Antisense GAT CCA CAC TCT CCA GCT GCA

IL6 Interleukin-6

Sense GAG GAT ACC ACT CCC AAC AGA CC

Antisense AAG TGC ATC ATC GTT GTT CAT ACA

Probe TCC TAC CCC AAT TTC CAA TGC TCT CC

IL17 Interleukin-17

Sense AGC AAG GAA TGT GGA TTC AGA G

Antisense CAG AAA AAC AAA CAC GAA GCA G

Probe TGC CCT CCA CAA TGA AAA GAA GGTG

iNOS inducible nitric oxide synthase

Sense CAG CTG GGC TGT ACA AAC CTT

Antisense CAT TGG AAG TGA AGC GTT TCG

Probe CGG GCA GCC TGT GAG ACC TTT GA

Pol2a RNA polymerase 2a

Sense ACC ACG TCC AAT GAT ATT GTG GAG

Antisense ATG TCA TAG TGT CAC ACA GGA GCG

Probe CTG GGC ATT GAG GCT GTG CGG AA

S100A8 Calgranulin A

Sense CTC CGT CTT CAA GAC ATC GTT TG

Antisense TCA TTC TTG TAG AGG GCA TGG TG

Probe CAA TGC CGT CTG AAC TGG AGA AGG CC

SPP1 Osteopontin

Sense GCT TGG CTT ATG GAC TGA GG

Antisense CCT CAT CTG TGG CAT CAG G

Probe TCA AAG TCT AGG AGT TTC CAG GTT TCT GAT GA

TNFα Tumor necrosis factor-α

(Continued)

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Preparation of Platelets for Intra-Vital Epifluorescence High SpeedVideo MicroscopyCitrated whole mouse blood was collected by intra cardial puncture. Murine platelets were iso-lated and labeled with 12.5 μg/mL 5-(and 6-) Carboxyflourescein diacetate, succinimidyl ester(Invitrogen Life Technologies GmbH; Carlsbad, California, USA) as reported earlier [20]. Thelabeled platelet suspension was adjusted to a final concentration of 150 x 103 platelets/μL and250 μL of the suspension was injected intra venously via a jugular vein catheter.

Intra-Vital Epifluorescence High Speed Video MicroscopyMeasurements were performed with a high-speed wide-field Olympus BX51WI fluorescence mi-croscope, using a long-distance condenser and a 10x (NA 0.3) water immersion objective with amonochromator (MT 20E; Olympus Deutschland GmbH; Hamburg, Germany) and a charge-coupled device camera (ORCA-R2; Hamamatsu Photonics; Hamamatsu, Japan). For image ac-quisition and analysis, the Realtime Imaging System eXcellence RT software (Olympus Deutsch-land GmbH) was used. Cell recruitment was quantified in one field of view (100 μm x 150 μm)per injury area. Adherent cells were defined as cells that did not move or detach from the endo-thelial lining within an observation period of 20 seconds and presented per mm2.

Isolation of GaliellalactoneGaliellalactone, a tetrahydro-isobenzofuranone derivative, from the ascomycete strain A111-95was isolated as previously described [12].

The ascomycete strain A111-95 was isolated from wood. The strain was kindly provided byH. Anke and is deposited in the culture collection of the LB Biotechnologie, University Kaisers-lautern. For maintenance on agar slants the strain was kept on YMGmedium composed of: yeastextract 0.4%, malt extract 1%, glucose 1%, pH 5.5 and agar 1.5% for solid media. Fermentationswere carried out in a Braun Biostat A-20 fermenter containing 20 liters of YMGmedium withaeration (3 l air/ min) and agitation (120 rpm) at 22°C. The production of galiellalactone was fol-lowed by the inhibitory effect of various concentrations of a crude extract of the culture fluid inthe IL-6-dependent reporter gene assays as described below. After 700 h of fermentation, the cul-ture fluid was separated from the mycelium by filtration and extracted with EtOAc. The solventwas evaporated and the crude product (2.3 g) was separated by chromatography on silica gel(Merck 60) with cyclohexane:EtOAc (70:30) as eluent resulting in 1.2 g of an enriched product.Preparative HPLC (Macherey-Nagel Nucleosil 100–7 C-18, column 40U250 mm) with water:MeOH (46:54) as eluent yielded 635 mg galiellalactone. The purity of the isolated compound asestimated by HPLC-DAD/MS analysis was greater than 98.5%. Gal was resolved in ethanol abs.and administered at a concentration of 10 mg/kg in PBS containing 10% ethanol.

StatisticsData represent means + SEM, as not stated otherwise. Statistical differences were determined byfactorial analysis of variance followed by one-way or two-way ANOVAmultiple comparison

Table 1. (Continued)

Sense CAT CTT CTC AAA ATT CGA GTG ACA

Antisense TGG GAG TAG ACA AGG TAC AAC CC

Probe CAC GTC GTA GCA AAC CAC CAA GTG G

doi:10.1371/journal.pone.0130401.t001

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test. In the case of two means, classic t-test analyses were used. All statistical analyses were per-formed using Graphpad Prism 5.0d.

Results

Galiellalactone Reduces Pro-Inflammatory Marker Gene Expression inPro-Atherosclerotic MiceTo test whether Gal-treatment results in reduced expression of pro-inflammatory genes in con-ductance arteries, we studied the expression profile of major pro-inflammatory mediators inthe aorta of mice onWD that were either treated with vehicle (PBS+EtOH) or Gal. When com-pared with the ND group, feeding ApoE-deficient mice with WD for 18 weeks and treatingthem for 6 weeks with vehicle (PBS+EtOH) led to an increase of most analyzed marker genemRNAs in the aorta that are known to have pro-atherosclerotic and/or pro-inflammatory ca-pacities (Fig 1). These results demonstrate a highly inflamed and atherosclerosis-prone statusin the aorta of WD- and vehicle-treated mice.

Gal-treatment of ApoE-deficient mice fed with WD for 18 weeks, decreased mRNA expres-sion of the marker genes when compared to mice treated with vehicle. Gal-administration sig-nificantly reduced mRNA expression levels of pro-inflammatory TNFα (Fig 1C) andinterleukin-17 (IL17) (Fig 1G), both published to be involved in atherosclerotic changes inApoE-deficient mice [22, 23], Calgranulin A (S100A8) that is a marker for chronic inflamma-tory diseases (Fig 1D) [24], as well as the atherogenesis-related marker Cathepsin S (CTSS;Fig 1A) [25]. Furthermore, Gal-treatment decreased the mRNA-expression of other markersup to certain levels: Osteopontin (SPP1; Fig 1B), known to recruit monocytes/macrophages[26], inducible nitric oxide synthase (iNOS) that is critical for many immunomodulatorymechanisms (Fig 1E), as well as the pro-inflammatory cytokine IL6 (Fig 1F).

As no significant induction of IL1β and CCL2 (chemokine C-C motif ligand 2, also namedmonocyte chemotactic protein 1—MCP1) mRNA expression was detected in the same aortaRNA preparations, (see S1 Fig), we consequently measured no effect of Gal on the expressionof these mRNAs.

Treatment with Galiellalactone Leads to Prolonged Blood Clotting TimesTo analyze whether Gal-treatment has functional effects on blood coagulation, which can induceplatelet activation and is essential to stabilize nascent thrombi, we performed coagulation assaysusing plasma of ApoE-deficient mice fed withWD and treated either with vehicle or Gal.

An every other day-treatment of mice with Gal over six weeks increases the time needed forcomplete coagulation of the samples (Fig 2). This increase was noted for both the intrinsic andthe extrinsic pathway of the coagulation cascade, as PT values represent the extrinsic and aPTTvalues the intrinsic and common pathway of coagulation.

To check if these changes in blood clotting times are related to changes in the expression ofclotting factors in the liver, we determined in qPCR analyses the mRNA expression of the clot-ting factors FII, FVIII and FX in the livers of the animals. As shown in S2 Fig, no effects of Galon the mRNA expression of these factors could be detected.

Galiellalactone Reduces Platelet Adhesion to Injured Arterial BloodVesselsTo analyze the anti-thrombotic capacity of Gal, we determined, whether a 6-week treatment ofpro-atherosclerotic ApoE-deficient mice either fed with ND or WD (treated with Gal or vehi-cle) has an impact on platelet adhesion to the ligation-injured carotid artery, a vascular side

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where atherosclerotic plaques frequently develop and rupture. To monitor platelet adhesionover the time, a common carotid artery thrombosis model was used, in which fluorescence-la-beled platelets isolated from a donor mouse of the same genotype and background were in-jected into the acceptor mouse [27]. Adhesion of fluorescent platelets was visualized in situ byintra-vital epifluorescence high-speed video microscopy.

Feeding the mice with WD for 18 weeks increased the adhesion of platelets to the injuredarea by two-fold (Fig 3 and S1–S3 movies) when compared to NDmice. Gal-treatment revertedthis increase to the level of adherent platelet counts of mice fed with ND.

To analyze whether Gal changes the numbers of the different blood cells, we performedblood cell counts. As seen in Fig 4, no effects of Gal on the blood cells counts in WD-fed ani-mals were detected.

Fig 1. Galiellalactone reduces the expression of pro-inflammatory marker genes in pro-atherosclerotic mice.RNA isolated from aortas of ApoE-deficient mice either fed for 18 weeks with normal chow diet (ND) or western-type diet (WD) (treated with Galiellalactone (Gal) or vehicle (PBS/EtOH) for 6weeks) was analyzed for CTSS (A), SPP1 (B), TNFα (C), S100A8 (D), iNOS (E), IL6 (F), and IL17 (G) mRNA expression in qPCR experiments. Data shownare mean + SEM of 4–10 mice (*** = p < 0.001; ** = p < 0.01; * = p < 0.05; ns = not significant vs. WD + PBS/EtOH; one-way ANOVA).

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DiscussionIncreased cardiovascular mortality of patients suffering from inflammatory diseases is relatedto enhanced atherosclerotic and thrombotic events that are independent of the classic risk fac-tors for cardiovascular death [6, 28]. Elevated inflammation seems to contribute to this en-hanced risk [7] and, therefore, new drugs that on the one hand are able to lower inflammationand on the other hand reduce the cardiovascular mortality by preventing arterial thrombus for-mation are needed.

Murine models with highly elevated pro-inflammatory gene expression (like Tristetrapro-lin-deficient mice [29]) have a complex disease phenotype and are not well suited for long-term analyses needed for cardiovascular research due to their markedly reduced life span. Thecardiovascular phenotype of such mice is generally insufficiently characterized. In contrast, theApoE-deficient mice are a well-established model of atherosclerosis. Moreover, the athero-thrombotic changes in ApoE-deficient mice have been correlated to enhanced inflammation[30, 31]. Therefore, selected this model to study the anti-inflammatory and anti-thrombotic ca-pacity of the fungal compound Gal [12]. Gal-treatment led to a reduction of WD-inducedmRNA expression of different inflammatory and atherosclerotic marker genes (Fig 1). Thoseresults indicate that Gal is able to lower at least the mRNA expression of genes that are involvedin atherogenesis and inflammation. As shown in suppl. Fig 1, we found no induction of IL1β orCCL2/MCP1 mRNA expression in the aortas of animals fed with WD compared to ND-treatedanimals. Contradictory data exist that show either an induction or no induction of IL1β andCCL2 expression by fat containing diets. In a gene expression profiling study by Castro et al[32] no enhanced IL1β or CCL2/MCP1 expression in the aorta of WD-fed ApoE-ko mice werefound. In contrast another type of high fat atherogenic diet increased IL1βmRNA expressionin the aortas of ApoE-ko mice [33]. In our study WD did not induce IL1β or CCL2/MCP1mRNA expression in the aorta of ApoE-ko mice. The inability of Gal to reduce IL1β and

Fig 2. Galiellalactone-treatment of pro-atherosclerotic mice leads to prolonged blood clotting times.Citrate-buffered plasma of ApoE-deficient mice fed with WD for 18 weeks and treated either with PBS/EtOHor Gal for the last 6 weeks was used for blood clotting studies in a coagulometer. PT values were obtained byadding Thromborel S, whereas aPTT values were obtained by addition of Pathromtin SL and calciumchloride. The time until a complete clotting of the sample occurred was measured. Data shown are mean+ SEM of 2–4 mice (* = p < 0.05 vs. PBS/EtOH-treated mice; t-test).

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CCL2/MCP1 mRNA expression in those aortas seems to be mainly contributed to an absenceof mRNA-inducibility of these genes by WD.

Coagulation activation and adhesion of platelets to the injured vessel wall are critical mecha-nisms for the progression of atherosclerosis and thrombosis [34, 35]. Treating atheroscleroticApoE-deficient mice with Gal significantly prolonged the blood clotting times of plasma ofthose mice in comparison to vehicle-treated mice (Fig 2). Enhanced blood clotting times maylower the thrombotic risk of the Gal-treated mice, as indicated in the applied platelet adhesionmodel at the ligation injured common carotid artery (Fig 3). Induction of the atheroscleroticphenotype by WD led to increased adhesion of platelets to the injured vessel wall that could berestored by Gal-treatment to the level of control mice (fed with ND). These changes are not re-lated to Gal-mediated changes in the blood cell counts as shown in Fig 4.

Fig 3. Galiellalactone reduces the adhesion of platelets to the ligation-injured arteria carotis communis. In a common carotid artery thrombosismodel, carotid injury in ApoE-deficient mice either fed for 18 weeks with normal chow diet (ND) or western-type diet (WD) (treated with Galiellalactone (Gal)or vehicle (PBS/EtOH) for 6 weeks) was induced for 5 minutes by transient ligation. The adhesion of fluorescence-labeled platelets of donor mice to theinjured blood vessel was observed every 5 minutes after injury in situ by high-speed intra-vital epifluorescence high-speed video microscopy for anobservation period of 30 minutes. The upper panel (A) shows representative images of adhering platelets 30 minutes after injury. Data shown in the lowerpanel (B) are mean ± SEM of 3–4 mice (*** = p < 0.001 vs. WD + PBS/EtOH; ns = not significant vs. ND; two-way ANOVA).

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Pro-inflammatory cytokines (e.g. IL1β, IL6, and TNFα), which are systemically enhanced inchronic inflammatory diseases like RA are described to trigger endothelial cells to change theiranti-thrombotic properties into a procoagulant state [36]. Gal seems to be able to reduce bloodclotting and platelet adhesion to the vessel wall by reduction of the inflammatory burden dueto its ability to interfere with different pro-inflammatory signaling pathways (like NF-κB- orSTAT-dependent gene expression).

In summary, the results of our study, using the fungal compound Gal for the first time in anatherosclerotic mouse model, showed that Gal is able to lower the expression of an inflamma-tory cytokine profile and improves key parameters of arterial thrombosis (blood clotting times,platelet adhesion to the ligation injured carotid artery). Therefore, Gal could be a promisingnew substance or lead-structure for the development anti-atherothrombotic drugs.

Supporting InformationS1 Fig. Western-type diet has no effect on IL1β or CCL2/MCP1 mRNA expression in theaorta of ApoE-deficient mice. RNA isolated from aortas of ApoE-deficient mice either fed for18 weeks with normal chow diet (ND) or western-type diet (WD) (treated with Galiellalactone

Fig 4. Galiellalactone has no effect on blood cell counts in western type diet-treated ApoE-deficient mice. ApoE-deficient mice were either fed for 18weeks with normal chow diet (ND) or western-type diet (WD) (treated with Galiellalactone (Gal) or vehicle (PBS/EtOH) for 6 weeks). Citrated whole mouseblood was collected by intra-cardial puncture. Platelet counts (A), white blood cell counts (WBCs;B) as well as red blood cell counts (RBCs;C) weredetermined using an automatic cell counter. Data shown are mean + SEM of 4–6 mice (ns = not significant vs. WD + PBS/EtOH; one-way ANOVA).

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(Gal) or vehicle (PBS/EtOH) for 6 weeks) was analyzed for IL1β (A) and CCL2/MCP1 (B)mRNA expression in qPCR experiments. Data shown are mean + SEM of 4–10 mice (ns = notsignificant vs. WD + PBS/EtOH; one-way ANOVA).(TIFF)

S2 Fig. Galiellalactone has no effect on the expression of blood clotting factors in the liverof western type diet-treated ApoE-deficient mice. RNA isolated from livers of ApoE-deficientmice either fed for 18 weeks with normal chow diet (ND) or western-type diet (WD) (treatedwith Galiellalactone (Gal) or vehicle (PBS/EtOH) for 6 weeks) was analyzed for factor II (FII;A), factor VIII (FVIII; B), and factor X (FX; C) mRNA expression in qPCR experiments. Datashown are mean + SEM of 4–10 mice (� = p< 0.05; ns = not significant vs. WD + PBS/EtOH;one-way ANOVA).(TIFF)

S1 Movie. Adhesion of platelets to injured vessel wall in NDmice. Imaging of 5-(and 6-)Carboxyflourescein diacetate succinimidyl ester stained adhering platelets (green) to the in-jured vessel wall of the common carotid artery in ApoE-deficient mice fed with normal chowdiet (ND) 30 min after transient ligature, movie of a representative sample.(AVI)

S2 Movie. Adhesion of platelets to injured vessel wall in WDmice treated with vehicle. Im-aging of 5-(and 6-) Carboxyflourescein diacetate succinimidyl ester stained adhering platelets(green) to the injured vessel wall of the common carotid artery in ApoE-deficient mice fed withwestern-type diet (WD) and treated with vehicle (PBS + 10% ethanol) 30 min after transientligature, movie of a representative sample.(AVI)

S3 Movie. Adhesion of platelets to injured vessel wall in WDmice treated with Gal. Imagingof 5-(and 6-) Carboxyflourescein diacetate succinimidyl ester stained adhering platelets (green)to the injured vessel wall of the common carotid artery in ApoE-deficient mice fed with west-ern-type diet (WD) and treated with the fungal metabolite Galiellalactone (Gal; 10 mg/kg) 30min after transient ligature, movie of a representative sample.(AVI)

AcknowledgmentsWe thank M. Göllner, I. Ihrig-Biedert, K. Masch, M. Michel, and G. Reifenberg (Departmentof Pharmacology, University Medical Center, Mainz, Germany) for their excellenttechnical support.

Author ContributionsConceived and designed the experiments: FB SJ CR KJ HL GE UWHK AP. Performed the ex-periments: FB SJ KJ ZW NX GE LS KS. Analyzed the data: FB SJ KJ ZWNX LS KS. Contribut-ed reagents/materials/analysis tools: SJ KJ HL GE HK AP. Wrote the paper: FB SJ CR KJ HLHK AP LS KS.

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